Imaging enhancer-promoter interaction dynamics during embryonic brain development During development, the vertebrate central nervous system is formed by partitioning proliferating primordial cell populations into compartments and gradually refining these populations into distinct functional sub-re- gions. Differentiation and organization of the various cell types in these regions are orchestrated by the expres- sion of regulatory genes in precise spatial and temporal patterns. Gene expression, in particular its transcrip- tion, is regulated primarily by enhancers, DNA sequences that activate transcription by interacting with gene promoters. While much of what we understand about gene regulation by enhancers has been gained through transgenesis assays and more recently through chromosome conformation capture and its next generation de- rivatives, much is still unknown about how enhancers interact with gene promoters to activate transcription, how dynamic and stable these interactions are, and how multiple enhancers interact with gene promoters and each other. Thus, our understanding of cis-regulation may be advanced with the ability to characterize en- hancer-promoter interaction dynamics. This project will develop the reagents and technologies to directly measure the interactions among enhancer and promoter sequences in the fgf8a locus. Fgf8a is a morphogen that regulates critical developmental processes including proliferation and positional identity and has been well characterized in the developing brain and neural crest. In Aim 1, sequence specific probes will be developed that do not interfere with normal fgf8a expression. In Aim 2, genetically encoded fluorescent protein variants will be targeted to precise enhancer and promoter sequences in the genome. High resolution, selective plane illumination microscopy with sub-diffraction-limited point source localization of the fluorophores will be used to measure relative 3D spatial coordinates of the labeled sequences over time in the developing embryonic ver- tebrate brain. This information will be used to better understand the dynamics and stability of enhancer-en- hancer and enhancer-promoter interactions and its relationships with target gene transcription.